The ultimate objective of this study is to elucidate more fully the nature of the cellular defects in blood and marrow in paroxysmal nocturnal hemoglobinuria (PNH) and to relate these to various in vivo and in vitro manifestations of PNH. We will extend previous studies involving continued assessment of hemolysis, iron metabolism, therapy with a variety of agents and the effects ff chronic hemolysis and thrombosis on organ function and structure. Laboratory investigations will be predominantly in two directions: (1) elucidation of the mechanism of the sucrose hemolysis test; and (2) the nature of the membrane defect in PNH. We propose that a structural change occurs in a serum protein in the serum-sucrose mixture, which allows normal serum protein(s) to behave like an "antibody" and thus initiate hemolysis by the EAC 1,4,2,3...9 pathway. We will attempt to separate this "active" protein component and to characterize it. Available evidence indicates that the PNH red cell defect involves membrane proteins. AChE activity on the single cell level will be further assessed using a modified hsitochemical staining technique. Normal and PNH red cells will be treated with drugs known to affect microtubules and cause rigidity of the cell membrane and then tested in various PNH hemolytic systems. Another possible explanation of the membrane defect in PNH is suggested by the presence of proteolytic enzymes in the red cell membrane. We will examine PNH red cells for the presence of abnormal amounts of such enzymes or increased susceptibility to their action. Emphasis will also be placed on thrombosis in PNH (esp., hepatic venous thrombosis in terms of platelet and coagulation abnormalities). One of our thesis is that thrombosis in the microvascular circulation in PNH brings about organ changes strikingly similar to (albeit milder than) those seen in sickle cell anemia.